The medical and scientific community has been intrigued at the potential for harnessing the immune system to combat disease pathogens. Scientists have learned that the immune system relies on a symphony of elevated, unique types of cells in the blood and tissues, governed by hormones, in particular, the interleukins. It is a continuing pursuit of medical research to direct the power of the immune system toward control of disease pathogens such as cancer cells, tumor cells, viral infection and infectious diseases. See Smith, Scientific American, 50-57 (March 1990).
The lymphokine Interleukin-2 (IL-2) has a wide scope of activity and plays a key role in immune regulation. In clinical trials, IL-2 alone has produced tumor responses in approximately 15 to 20% of the patients with renal cell carcinoma and metastatic melanoma. Although IL-2 alone can mediate an anti-tumor response, it has limitations and is accompanied by severe toxicities in most current modalities. See Smith, op. cit.; Sznol, Seminars in Oncology 20:1, 80-93 1993); Cetus Oncology Div & Chiron Corp, IL-2 Brochure, 3-48 (1992); Devita, Hellman & Rosenberg, Cancer Principles and Practice of Oncology 1, 332-341 (1989); Haskell, Cancer Treatment, 3rd ed., 102-119, (1990); Stein & Dalgleish, European Journal of Cancer 30A:3, 400-404 (1994); Rosenberg & Yang et al., JAMA 271:12, 907-913 (1994).
In order to improve and potentiate the effect of IL-2 and lessen the toxicities associated with IL-2 treatment, effector cell transfer and expansion techniques have been developed. In a procedure known as adoptive cellular immunotherapy, effector lymphocytes are taken from the patient ("rescued") and expanded in in vitro cultures with IL-2. These lymphokine-activated killer (LAK) cells are then re-infused into the patient in conjunction with IL-2. Essentially these LAK cells are additional effector cells which are reinfused into the patient. This combination of additional effector (LAK) cells and IL-2 results in more effective therapy than IL-2 alone.
Although the addition of these effector cells resulted in a trend toward more long term survivors, the results were further improved upon by the continuing development of adoptive immunotherapy. Improved cell transfer and expansion techniques made possible the rescue, removal and expansion in vitro of more tumor specific effector cells from patients with cancer, which in animal tumor models were 100 times as potent as previously used LAK cells on a per cell basis. See Devita, Hellman & Rosenberg, op. cit.; Rosenberg et al, Journal of the National Cancer Institute 86:15, 1159-1166 (1994); Haskell, op. cit.; Stein, op. cit.; Rosenberg & Yang, JAMA, op. cit. These improved effector cell transfer and expansion techniques involve the growth of tumor-infiltrating lymphocytes (TILS) by first extracting bits of tumor from the patient (rescue), then expanding the effector cells therein in vitro, followed by re-infusing the expanded effector cells into the patient. Initial treatments may be followed by further growing of more effector TILS for a second and third course of treatment.
However, there are substantial limitations to this present state of the art technique. The limitations are that it is very expensive in terms of time, equipment and consumables. Further, the original effector TIL cells can only be rescued from a fraction of cancer patients and many tumor sites are inaccessible to this approach. Even if original effector TILS are rescued from a tumor site, only approximately 50 to 70% can be expanded in vitro to provide a suitable dose for treatment. In addition, the actual growing of these TILS is very complex and difficult. Each cancer patient's cells react differently to in vitro effector cell transfer techniques. Even after the first set of TILS are rescued and expanded in vitro, the second or third set for future treatment may not grow. Also, the very invasiveness of extracting tumor from a patient (rescue) could have a tendency to spread tumor cells in the blood system, thus possibly promoting and spreading tumor growth.
Moreover, because the time and labor required to grow these cells for the majority of patients is 20 to 60 days, it is burdensome and time consuming for the medical team. Since each patient's effector TILS grow differently, the rate of growth of each patient's effector cells affects the anti-tumor activity of the cells. Namely, the slower growing TILS have less anti-tumor activity than the more quickly growing effector TILS. Furthermore, the actual administration of the effector TIL cells, in two to four separate bags, separated by several hours, is burdensome and invasive to the patient and requires massive amounts of IL-2 for further activation. The procedure is associated with a grade 3 or 4 toxicity, which means admittance to Intensive Care Unit (ICU) (see Rosenberg, JNCI, op. cit.).
In sum, although the addition of these TIL effector cells is more potent than IL-2 alone or IL-2 with LAK effector cells, this technology, with its complexity, cost, invasiveness, time and other aforementioned aspects, does not lend itself to the American Community Hospitals. Also, the addition of TILS is only one type of effector cell, it does not address other potent effector cells such as NK cells, macrophages, B cells, Neutrophils (PMNs) and others, which are all responsive to and activated by the lymphokine IL-2 to mediate an anti-tumor response. For instance, IL-2 treated patients demonstrate a T cell and macrophage inflammatory infiltrate at tumor sites. The immune system operates as a symphony with myriad complexity and interactions with a multitude of effector cells. This effector TIL technique does not address this.
The lymphokine IL-2 exerts its effects through a specific saturable receptor system, located primarily on T cells, B cells, NK cells and LAK cells. The bulk of the IL-2 dependent activity occurs via the high affinity receptor. The effector cell transfer and expansion TIL technique does not address or direct its activity toward the high affinity IL-2 receptor. Therefore, even if these TIL effector cells are re-infused in the patient, these same effector TIL cells would not be any more receptive to IL-2 because nothing has been done to up-regulate their high affinity IL-2 receptors.
Currently under investigation is the utilization of monoclonal antibodies to stimulate and/or enhance host immune effector mechanisms along with several other techniques. See Nakajima et al., Proc. Natl. Acad. Sci. USA 91, 7889-7893 (1994). Another technique currently being investigated is the insertion of the gene for the high affinity IL-2 receptor in effector TILS. This technique is exceedingly complex, expensive, difficult and does not appear to work in sufficient amount of cells to be clinically relevant at this time. The grade 3 and/or 4 toxicity associated with IL-2 and effector TILS treatment is a severe detriment to this method of increasing effector cells and potentiating lymphokine IL-2 activity.
In a study of IL-2 and TILS, effector TILS were characterized extensively. See Rosenberg et al., op. cit. These TILS, which were rescued from a tumor site, expanded in vitro, and re-infused in the patient were primarily CD-3+, CD-4+, CD-8+ and CD-56. The sole advantage of effector TIL rescue, expansion and re-infusion is that patients treated with IL-2 alone demonstrated a 17% response rate; whereas the addition of effector TILS increased the response rate to 34%, indicating that the addition of effector cells adds therapeutic benefit. See Cetus, op. cit.; Devita, Hellman & Rosenberg, op. cit.; Rosenberg et al., JNCI, op. cit. Further evidence that the addition of effector cells, such as TILS, adds therapeutic benefit to lymphokine administration is that some patients who have failed lymphokine, for instance, IL-2, administration alone, respond to a subsequent course of IL-2 plus effector cells. These results suggest that the addition of effector cells to lymphokine administration adds therapeutic benefit.
The present invention is directed to a method for adding and maintaining effector cells prior to and during treatment to potentiate and increase therapeutic benefit derived from the administration of a lymphokine, such as IL-2, or IL-12, a cytokine, biological response modifier and/or pharmacological agents directed toward disease pathogens. The present invention appears to cause a broad based leukocytosis, lymphocytosis and increase of effector cells in vivo, and to increase the same sub-population of lymphocytes as the aforementioned effector TILS in vivo and in an apparently essentially non-toxic to minimally toxic manner.
It is generally believed that the larger the population of effector cells, the better the chance for a favorable resolution for many disease states, including cancer, and viral, bacterial and protozoa infection. Historically and currently this increase in effector cells is known as an active non-specific response. Examples of agents which have been used in this modality are: Bacille Calmette-Geurin--methanol extractable residue (BCG-MER), Coryne bacterium parvum, levamisole, thymosines, and Coleys mixed toxins. Results of studies of these agents show little value and a checkered history because it apparently is inadequate to simply expand the quantity of effector cells alone without administering a lymphokine, cytokine and/or biological response modifier in order to activate and turn these effector cells on to elicit a cytotoxic anti-pathogen response from these cells.
These methods of increasing effector cells have substantial shortcomings. Among the most serious are production of antibodies and tolerance. See Devita, Hellman & Rosenberg, Ch 17, op. cit.; Haskell, op. cit. The aforementioned agents all engender the creation of specific antibodies and the patient subsequently develops irreversible tolerance to these agents. Therefore, administration of these agents will apparently not elevate effector cells after a series of administrations. Furthermore, since antibody is created, the bulk of increase of effector cells with little activity is directed toward the reaction to these agents and only the excess immune cells (by-standers) are available to fight the disease pathogen or be stimulated by subsequent lymphokine/cytokine administration. These agents also produce their own unique toxicities. For instance, BCG is known to have substantial toxicities associated with its use. See Devita, Hellman & Rosenberg, Ch 17, op. cit.; Haskell, op. cit.
None of the aforementioned methods, including adoptive immunotherapy, are known to induce the expression of the high affinity IL-2 receptor on effector lymphocytes. Tanaka et al. (Scand. J. Immuno. 31, 547-552 1990), have shown that the specific culturing in vitro of human peripheral blood mononuclear cells with high concentrations of zinc induces the expression of high affinity IL-2 receptors on effector lymphocytes. The present invention raises the intracellular concentration of zinc in whole blood thus producing in vivo, the in vitro conditions reported by Tanaka. It is believed that this induces the expression of high affinity IL-2 receptors on lymphocytes. See Hamdi, Brit. J. Indust. Med. 26, 126-134 (1969).
The effect of zinc exposure by ingestion, inhalation or other means on human health and safety has been extensively studied. Zinc oxide fume inhalation, in every reference to it from pre-christian times onwards, is considered a nuisance, occupational disease and an industrial toxicological problem. See NIOSH, criteria document. There does not appear to be any suggestion of possible or potential medical usefulness in the historical reports to date.
The present invention involves administration of zinc by zinc oxide fume inhalation. It is believed that effects equivalent to the present invention are not achievable by other forms of administration of zinc, including ingestion of up to and including toxic levels in humans, since sufficient elevation of intracellular zinc levels and symptomology of zinc metal fume fever including expansion of effector cells is not thereby attained. Studies have shown that up to 300 mg of zinc, 10 fold more than is administered by inhalation by the present invention, administered orally does not elevate the intracellular level of zinc or expand effector cells. Study suggests that excess ingested zinc is stored in the plasma without expansion of effector cells. See Chandra, JAMA 252:11, 1443-1446 (1984).
The safety of zinc oxide fume inhalation has been ascertained by agencies of the United States Government and by published case reports and experimental studies. See Blanc, Annals of Internal Medicine 114, 930-936, 1991; Drinker et al., Journal of Industrial Hygiene 9:8, 88-97, 98-108, 331-345, 1927; NIOSH, Recommended Standard, US Dept. of Hlth, Educ, & Welfare, 657-645/220, 1976; NIOSH, National Institute for Occupational Safety, Criteria Document PB-246-693, 1-52 1975; Occupational Hlth Guideline for Zinc Oxide Fume, US Dept of Hlth & Human Svc--US Dept of Labor, Sept 1978; National Safety Council, Data Sheet 1-267-Rev 86. As an example of the lack of toxicity of the present invention, Hamdi (op. cit.) reports on page 127, that the 12 workers, working 8 hours a day, 6 days a week with heavy exposure to zinc oxide fume, exposed several times a day from 4 to 21 years, with the average duration of exposure of 11 years, demonstrated no apparent toxicity.
Zinc oxide fume inhalation according to the present invention is directly useful in potentiating existing and emerging treatments in cancer medicine, AIDS and AIDS-related complex (ARC) where the patient would benefit in an increase in effector cells and up-regulation of lymphokine IL-2 receptors on these same cells in preparation for lymphokine and cytokine administration. AIDS is characterized by progressive destruction of CD-4 lymphocytes. In addition it has been discovered that CD-8 lymphocytes are very useful in combating HIV disease and holding the virus in check. The present invention would expand effector CD-4 and effector CD-8 populations in afflicted patients and up-regulate the IL-2 receptor on these same cells in preparation for administration of cytokines, such as the lymphokine IL-2.
Moreover, IL-12, a potent lymphokine, activates natural killer cells. Natural killer cells have been shown in vitro, in the presence of IL-12, to correct some of the immunological deficiencies of blood taken from HIV patients and to kill the AIDS virus in vitro. See Stein, op. cit.; Cancer Research Institute 1:2, 1-8, 1994; J. of Experimental Medicine, May 1992, Science, Dec. 10, 1993, Science, Oct. 8, 1993.
In cancer and HIV medicine, the expansion and up-regulation of all these distinct populations of effector cells which respond to IL-2, IL-12 or other lymphokines or cytokines is directly useful in increasing existing therapeutic response and extending indications for the usefulness of lymphokines and cytokines.
The agent itself, in addition to expanding effector cells and up-regulating high affinity IL-2 receptors, synergizes with lymphokines and cytokines, such as IL-2 and potentiates the effect of IL-2. See Tanaka, op. cit. The present invention addresses the shortcomings and of the current adoptive immunotherapy modality used in preparation for administration of lymphokines, including IL-2 and IL-12, and cytokines, including the interferons. See Stein et al., op. cit.; Nakajima et al., op. cit.
Among the advantages of the present invention are that it does not appear to produce tolerance other than tachyphylaxis. Thus no tolerance is developed to successive treatments. Accordingly, long term and short term treatment is feasible without chronic or acute toxicity. See Niosh, criteria doc; Occupational Health Guideline, op. cit.
It is thus an object of the present invention to provide a relatively simple, nontoxic and nonspecific method of increasing and maintaining immune effector cells in vivo and up-regulating high affinity IL-2 receptors on such cells.
In particular, it is an object of the present invention to provide a method of increasing the quantity and quality of effector cells and IL-2 receptors in patients afflicted with cancer, or viral, bacterial, or other infectious diseases or pathogens, before and during treatment with lymphokines, including IL-2, or other cytokines, biological response modifiers or pharmacological immunomodulators.
Further, it is an object of the present invention to provide a method of increasing the quantity and quality of immune effector cells in patients afflicted with cancer, viral, bacterial, or other infectious diseases or pathogens, in preparation and maintenance of such effector cells before and during treatment with lymphokines, including IL-2 and IL-12, cytokines, biological response modifiers and other pharmacological immunomodulators. Such immune effector cells include T cells (T-11), B cells (B-1), mature t cells (T-3), T helper/inducer (T-4), T suppressor/cyto (T-8), natural killer (NK), macrophages, neutrophils (PMNs) and other leukocytes.